The factors and cellular interactions that influence the commitment of cells to specific neural lineages are not well understood. We have used cultured non-neuronal process-bearing (PB) cells from neonatal rat cerebral cortices as a model to assess the influence of various culture conditions on the determination of cells as either astroglia or oligodendroglia. Increasing postseparation plating density was significantly associated (p less than 0.001) with decreasing percentages of glial fibrillary acidic protein (GFAP+) cells, increasing percentages of galactocerebroside (GC+) cells, and increasing percentages of nonstained cells. As the fetal calf serum content of growth medium was increased, the percentage of GFAP+ cells increased, and as the serum content was decreased, the percentage of GC+ cells increased. Evidence of minimal cell proliferation and the observation of PB cells that coexpressed GFAP and GC supported the conclusion that PB cells switched their phenotypic expression from GFAP+ in serum to GC+ in serum-free medium. PB cells exhibited plasticity in their phenotypic expression as cells grown for 9 d in serum-free medium were still responsive to the effects of serum, while cells grown for 6 d in serum were refractory to serum withdrawal. This research has demonstrated the plasticity of PB cells separated from polygonal astroglia as they expressed GFAP in the presence of serum and GC in serum-free medium.